Fig. 7

Autophagy inhibitor accelerates proteasome-dependent degradation of Nrf2 in mice. A Schematics of the in vivo experimental workflow. C57BL/6 J mice were treated with vehicle (PBS) or PBS diluted 3-MA (15 mg/kg) for 2 h via intraperitoneal injection (i.p.), and then exposed to CuONPs via intratracheal instillation (i.t.) for 3 days. B, C Mice were instilled intratracheally with CuONPs for 3 days. Immunoblotting analysis and quantification of protein levels of Sqstm1, LC3b and β-actin (loading control) in mice aorta tissues. D, E Mice were pretreated intraperitoneally with or without 3-MA and then instilled intratracheally with CuONPs for 3 days. Immunoblotting analysis and quantification of protein levels of Ubiquitin, Nrf2, Hmox1, Sqstm1 and β-actin (loading control) in mice aorta tissues. F Representative images of immunohistochemistry using antibodies against MMP-2 in the intima and media region of abdominal aorta. Black arrows indicate high expression regions of MMP-2. Scale bar, 50 μm. G The mRNA expression levels of Il6, Edn1 and Selplg in mouse aorta. In C, Student’s t-test was used for statistical significance. In E and G, one-way ANOVA followed by a Tukey multiple comparison test was used for statistical significance. All data are representative of three independent experiments. The values are expressed in mean ± S.D. “*”, P ≤ 0.05; “***”, “P ≤ 0.001”